A plasma membrane glycoprotein, present on normal eukaryotic cells, but missing or greatly reduced following transformation, has been shown to mediate adhesion of cells to collagen substratum. In accordance with this biological activity the protein has been designated Cell Adhesion Factor (CAF). We have now established that CAF specifically mediates adhesion and spreading of fibroblasts in an energy-dependent fashion, but independent of de novo protein synthesis. Microtubles and microfilaments are required for spreading but not attachment. CAF has not affinity for the cell surface unless it is first complexed to collagen implying that an activation step is necessary for CAF activity. Transformed cell migration can be reduced by the addition of 125 microns g/ml exogenous CAF. Cyanogen bromide cleavage of CAF generates a fragment of 45,000 daltons which retains the collagen binding site. The fragment, representing 10 percent of the intact molecules, also contains 30-50 percent of the total half cysteines present in CAF. Removal of carbohydrate, reduction and alkylation, oxidation of tryptophan with NBS, and citraconylation have no effect on CAF biological activity. Performic acid oxidation and treatment with DNFB both disrupt CAF function.